SINE RNA of the imprinted miRNA clusters mediates constitutive type III interferon expression and antiviral protection in hemochorial placentas.

Hemochorial placentas have evolved defense mechanisms to prevent the vertical transmission of viruses to the immunologically underdeveloped fetus. Unlike somatic cells that require pathogen-associated molecular patterns to stimulate interferon production, placental trophoblasts constitutively produce type III interferons (IFNL) through an unknown mechanism. We demonstrate that transcripts of short interspersed nuclear elements (SINEs) embedded in miRNA clusters within the placenta trigger a viral mimicry response that induces IFNL and confers antiviral protection. Alu SINEs within primate-specific chromosome 19 (C19MC) and B1 SINEs within rodent-specific microRNA cluster on chromosome 2 (C2MC) produce dsRNAs that activate RIG-I-like receptors (RLRs) and downstream IFNL production. Homozygous C2MC knockout mouse trophoblast stem (mTS) cells and placentas lose intrinsic IFN expression and antiviral protection, whereas B1 RNA overexpression restores C2MCΔ/Δ mTS cell viral resistance. Our results uncover a convergently evolved mechanism whereby SINE RNAs drive antiviral resistance in hemochorial placentas, placing SINEs as integral players in innate immunity.

The vanRCd Mutation 343A>G, Resulting in a Thr115Ala Substitution, Is Associated with an Elevated Minimum Inhibitory Concentration (MIC) of Vancomycin in Clostridioides difficile Clinical Isolates from Florida.

Clostridioides difficile, the primary cause of nosocomial antibiotic-associated diarrhea, has a complex relationship with antibiotics. While the use of broad-spectrum antibiotics disrupts the gut microbiota and increases the risk of C. difficile infection (CDI), antibiotics are also the primary treatment for CDI. However, only a few antibiotics, including vancomycin, fidaxomicin, and rifaximin, are effective against CDI, and resistance to these antibiotics has emerged recently. In this study, we report the identification of two RT027 C. difficile clinical isolates (TGH35 and TGH64) obtained from symptomatic CDI-diagnosed patients in Tampa, Florida in 2016. These two strains showed an elevated minimum inhibitory concentration (MIC) of vancomycin (MIC = 4 µg/mL, compared to the EUCAST breakpoint of 2 µg/mL) and contained a vanRCd 343A>G mutation resulting in a Thr115Ala substitution in the VanRCd response regulator. This mutation was absent in the vancomycin-sensitive control epidemic strain RT027/R20291. TGH64 was also resistant to rifaximin (MIC ≥ 128 µg/mL) and carried the previously reported Arg505Lys and Ile548Met mutations in RpoB. Furthermore, we report on the antimicrobial resistance (AMR) and genomic characterization of additional C. difficile isolates, including RT106/TGH120, RT017/TGH33, and RT017/TGH51, obtained from the same patient sample cohort representing the highly prevalent and regionally distributed C. difficile ribotypes worldwide. Considering that the VanRCd Thr115Ala mutation was also independently reported in seven C. difficile clinical isolates from Texas and Israel in 2019, we recommend epidemiological surveillance to better understand the impact of this mutation on vancomycin resistance. IMPORTANCE The perpetually evolving antimicrobial resistance (AMR) of C. difficile is an important contributor to its epidemiology and is a grave concern to global public health. This exacerbates the challenge of treating the infections caused by this multidrug-resistant causative organism of potentially life-threatening diarrhea. Further, the novel resistance-determining factors can be transferred between different strains and species of bacteria and cause the spread of AMR in clinical, environmental, and community settings. In this study, we have identified a mutation (vanRCd 343A>G) that causes a Thr115Ala substitution and is linked to an increased MIC of vancomycin in clinical isolates of C. difficile obtained from Florida in 2016. Understanding the mechanisms of AMR, especially those of newly evolving strains, is essential to effectively guide antibiotic stewardship policies to combat antibiotic resistance as well as to discover novel therapeutic targets.

Draft Genome Sequences and Genome Characterization of Three Toxigenic and Two Nontoxigenic Clostridioides difficile Clinical Isolates from Florida, USA.

Draft genome sequences of five Clostridioides difficile clinical isolates were obtained in Florida, USA. Three isolates, designated TGH29 (sequence type 1 [ST1]/clade 2), TGH79 (ST11/clade 5), and TGH91 (ST35/clade 1), contained toxin-encoding genes. The two nontoxigenic strains were classified as TGH114 (ST109/clade 4) and TGH132 (ST15/clade 1). Antimicrobial resistance determinants and plasmids were detected and putative prophages predicted in some isolates.

Preeclampsia is Associated With Reduced ISG15 Levels Impairing Extravillous Trophoblast Invasion.

Among several interleukin (IL)-6 family members, only IL-6 and IL-11 require a gp130 protein homodimer for intracellular signaling due to lack of intracellular signaling domain in the IL-6 receptor (IL-6R) and IL-11R. We previously reported enhanced decidual IL-6 and IL-11 levels at the maternal-fetal interface with significantly higher peri-membranous IL-6 immunostaining in adjacent interstitial trophoblasts in preeclampsia (PE) vs. gestational age (GA)-matched controls. This led us to hypothesize that competitive binding of these cytokines to the gp130 impairs extravillous trophoblast (EVT) differentiation, proliferation and/or invasion. Using global microarray analysis, the current study identified inhibition of interferon-stimulated gene 15 (ISG15) as the only gene affected by both IL-6 plus IL-11 vs. control or IL-6 or IL-11 treatment of primary human cytotrophoblast cultures. ISG15 immunostaining was specific to EVTs among other trophoblast types in the first and third trimester placental specimens, and significantly lower ISG15 levels were observed in EVT from PE vs. GA-matched control placentae (p = 0.006). Induction of primary trophoblastic stem cell cultures toward EVT linage increased ISG15 mRNA levels by 7.8-fold (p = 0.004). ISG15 silencing in HTR8/SVneo cultures, a first trimester EVT cell line, inhibited invasion, proliferation, expression of ITGB1 (a cell migration receptor) and filamentous actin while increasing expression of ITGB4 (a receptor for hemi-desmosomal adhesion). Moreover, ISG15 silencing further enhanced levels of IL-1ß-induced pro-inflammatory cytokines (CXCL8, IL-6 and CCL2) in HTR8/SVneo cells. Collectively, these results indicate that ISG15 acts as a critical regulator of EVT morphology and function and that diminished ISG15 expression is associated with PE, potentially mediating reduced interstitial trophoblast invasion and enhancing local inflammation at the maternal-fetal interface. Thus, agents inducing ISG15 expression may provide a novel therapeutic approach in PE.

Genomic and Phenotypic Characterization of the Nontoxigenic Clostridioides difficile Strain CCUG37785 and Demonstration of Its Therapeutic Potential for the Prevention of C. difficile Infection.

Symptoms of Clostridioides difficile infection (CDI) are attributed largely to two toxins, TcdA and TcdB. About 17-23% of C. difficile isolates produce binary toxin, which enhances C. difficile pathogenesis. Previously, we engineered the nontoxigenic C. difficile strain CCUG37785 (designated as CCUG37785) to express immunogenic fragments of TcdA and TcdB as an oral mucosal CDI vaccine candidate. In this study, we performed genomic and phenotypic analyses of CCUG37785 and evaluated its potential use for preventing and treating CDI. Whole genome sequencing showed that CCUG37785 is ribotype ST3 and lacks toxin genes. Comparative analyses of PaLoc and CdtLoc loci of CCUG37785 revealed 115-bp and 68-bp conserved fragments in these regions, respectively. Phenotypic comparisons between CCUG37785 and C. difficile R20291 (an epidemic hypervirulent BI/NAPI/027 strain, designated as R20291) found that CCUG37785 exhibited significantly higher adhesion and sporulation, significantly lower spore germination and biofilm formation, and comparable motility to R20291. We also showed that oral inoculation of CCUG37785 spores prior to infection with R20291 spores provided mice almost full protection against developing CDI. However, oral inoculation of CCUG37785 spores after infection with R20291 spores only provided minor protection against CDI. Further analysis showed that mice pretreated with CCUG37785 spores secreted significantly less R20291 spores, while mice treated with CCUG37785 spores after infection with R20291 secreted a comparable amount of R20291 spores to mice infected with R20291 spores only. Our data both highlight the potential use of CCUG37785 for the prevention of primary and recurrent CDI in humans and support its use as an oral mucosal vaccine carrier against CDI. IMPORTANCE Clostridioides difficile infection (CDI) symptoms range from diarrhea to intestinal inflammation/lesion and death and are mainly caused by two exotoxins, TcdA and TcdB. Active vaccination provides the attractive opportunity to prevent CDI and recurrence. No vaccine against CDI is currently licensed. Tremendous efforts have been devoted to developing vaccines targeting both toxins. However, ideally, vaccines should target both toxins and C. difficile cells/spores that transmit the disease and cause recurrence. Furthermore, C. difficile is an enteric pathogen, and mucosal/oral immunization would be particularly useful to protect the host against CDI considering that the gut is the main site of disease onset and progression. Data in our current study not only highlight the potential use of CCUG37785 to prevent primary and recurrent CDI in humans but also further support its use as an oral mucosal vaccine carrier against CDI.

Mechanisms of antibiotic resistance of Clostridioides difficile.

Clostridioides difficile (CD) is one of the top five urgent antibiotic resistance threats in USA. There is a worldwide increase in MDR of CD, with emergence of novel strains which are often more virulent and MDR. Antibiotic resistance in CD is constantly evolving with acquisition of novel resistance mechanisms, which can be transferred between different species of bacteria and among different CD strains present in the clinical setting, community, and environment. Therefore, understanding the antibiotic resistance mechanisms of CD is important to guide optimal antibiotic stewardship policies and to identify novel therapeutic targets to combat CD as well as other bacteria. Epidemiology of CD is driven by the evolution of antibiotic resistance. Prevalence of different CD strains and their characteristic resistomes show distinct global geographical patterns. Understanding epidemiologically driven and strain-specific characteristics of antibiotic resistance is important for effective epidemiological surveillance of antibiotic resistance and to curb the inter-strain and -species spread of the CD resistome. CD has developed resistance to antibiotics with diverse mechanisms such as drug alteration, modification of the antibiotic target site and extrusion of drugs via efflux pumps. In this review, we summarized the most recent advancements in the understanding of mechanisms of antibiotic resistance in CD and analysed the antibiotic resistance factors present in genomes of a few representative well known, epidemic and MDR CD strains found predominantly in different regions of the world.

CYP2D6 polymorphisms may predict occurrence of adverse effects to tamoxifen: a preliminary retrospective study.

INTRODUCTION AND AIMS: Tamoxifen is an adjuvant drug effective in treating hormone receptor - positive breast cancer. However, 30%-50% of patients relapse and many develop adverse effects, such as hot flashes and fatty liver. Allelic variations altering the activity of cytochrome P450-2D6 enzyme affect response to tamoxifen by modulating metabolism of tamoxifen into its pharmacologically active metabolite endoxifen. Although association between CYP2D6 polymorphisms and recurrence of breast cancer in patients on tamoxifen had been reported, little evidence exists on association between these polymorphisms and adverse effects to tamoxifen. This study explored the association between CYP2D6 polymorphisms and tamoxifen effects, hitherto not studied in Sri Lanka. METHODS: A retrospective preliminary study was carried out on 24 breast cancer patients on tamoxifen for minimally 3 months attending National Cancer Institute, Maharagama, Sri Lanka. They were not on CYP2D6-inhibiting drugs, chemotherapy or other endocrine therapy, and had no conditions that could occur as adverse effects to tamoxifen before starting the therapy. Their blood samples were collected, DNA was extracted and genotyped using SNaPshot Multiplex sequencing based single-nucleotide polymorphism (SNP) assay. RESULTS: SNP/allele frequencies detected: 1846G>A (confirmatory of *4 null allele)=8.3%; 2549delA (confirmatory of *3 null allele)=50%; 100C>T (suggestive of *10 reduced functional allele, in addition to other alleles)=0%; combination of 2988G>A, -1584C and 2850C>T (strongly suggestive of *41 or other reduced functional allele)=4.8%. Occurrence of heterozygous 2988G>A SNP with -1584C and 2850C>T was significantly higher among those with ultrasound-diagnosed fatty liver following the commencement of tamoxifen therapy (P=0.029). Adverse effects occurred at a significantly higher frequency among postmenopausal women (P=0.041). Three patients who developed recurrence of breast cancer had no association with SNPs tested. CONCLUSIONS: CYP2D6 SNP combination 2988G>A, -1584C and 2850C>T, strongly suggestive of *41 reduced functional allele, is likely to be useful in predicting occurrence of adverse effect fatty liver in breast cancer patients on tamoxifen, thereby alternative treatment can be considered and lifestyle modifications implemented. Larger sample studies are recommended with the measurement of tamoxifen and metabolite levels. Alternative therapy should be considered for postmenopausal patients.